作　　者： ; ; ; ; ; ; ;

机构地区： 暨南大学生命科学技术学院广东省生物工程药物重点实验室

出　　处： 《中国现代医学杂志》 2009年第22期3374-3377,共4页

摘　　要： 目的构建人巨细胞病毒UL49基因的诱饵表达载体,以利于筛选与pUL49相互作用的蛋白。方法PCR扩增UL49全序列,克隆入pGBKT7,将构建好的pGBKT7-UL49转化到酵母细胞AH109;用蛋白印迹法分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应。结果克隆成功pGBKT7-UL49;pG-BKT7-UL49成功转化到AH109,转化细胞AH109[pGBKT7-UL49]表达诱饵蛋白pUL49,pUL49对转化细胞无细胞毒性、无自激活。结论成功构建了酵母诱饵表达载体pGBKT7-UL49,为进一步筛选与pUL49相互作用的蛋白提供了实验基础。 [ Objective ] To construct the bait expression vector pGBKT7-UL49 of human cytomegalovirus UL49 gene coding protein for screening the target proteins interacting with the bait protein pUL49 through the yeast twohybrid system. [Methods] The fragments of UIA9 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKTT-UL49, being verified by sequencing, was transformed into AH109 yeast cells. Then the bait protein pUL49 was analyzed by Western Blotting. Toxicity and self-activation of the bait protein were detected by cultured in different SD. [ Results ] UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH109 as well and those cells exhibited neither toxicity nor self-activation. The expression of the bait protein pUL49 was detected by Western Blotting. [ Conclusion ] The bait expression vector of UL49 was constructed successfully, which layed the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique.

关 键 词： 酵母双杂交 人巨细胞病毒 质粒构建 诱饵蛋白

领　　域： [生物学]